Mechanisms involved in the formation of biocompatible lipid polymeric patchy particles

ABSTRACT

The invention relates to lipid polymeric patchy particles formed by nanoprecipitation and emulsification processes, utilizing a polymer blend including the polymer, solvent and lipid-PEGylated functional groups. More particularly, the invention relates to synthesizing particles having different or pre-selected morphologies (internal and external) and physicochemical properties. It has been found that the shear stress experienced by the polymer blend during emulsification can impart certain external and internal morphology and physicochemical properties to the resulting particles. Further, the one or more patches of the particles can be functionalized, such as, with gold nanoparticles, for use of the particles, in particular, in photoacoustic and ultrasound imaging.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Patent Application Ser. No. 62/120,984, filed Feb. 26, 2015,entitled “Mechanisms Involved in the Formation of Biocompatible LipidPolymeric Patchy Particles”, which is herein incorporated by reference.

GOVERNMENT SUPPORT

This invention was made with government support under Grant No.CBET-1348112 awarded by the National Science Foundation (NSF). Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

The invention relates to lipid polymeric patchy particles and synthesesfor their preparation. More particularly, the invention relates tomethods of controlling or tuning physicochemical properties and, theexternal and internal morphology, of the patchy particles.

BACKGROUND OF THE INVENTION

Patchy particles are a class of anisotropic particles that arecharacterized by having one or more surface-exposed domains withdifferent surface chemistry relative to the rest of the particle. Theanisotropic feature of nano- and micron-size patchy particles can becreated using several techniques including template-assistedfabrication, electrified jetting, glancing angle deposition,lithography, and phase segregation. The anisotropic feature isadvantageous because it allows fabrication of systems at nano- andmicron-scale sizes that can perform multiple functions. The patchypolymeric particles provide the ability to simultaneously present twodifferent surface chemistries on the same particle. There are diversemedical and industrial applications that may benefit from the use ofpatchy-lipid polymer particles including vaccines, drug delivery,sensors, photonics, imaging, tissue engineering and environmentchemistry.

There is a need in medicine for the development of theranostic devices,which are systems that perform at the same time therapeutic and imagingfunctions. In cancer, theranostic devices are needed because they can beused to distribute a drug homogenously in the tumor vasculature byassistance of an imaging function. In this way, the multi-drugresistance phenomenon, which is often observed in cancer, may besignificantly diminished. In tissue engineering, a patchy surface canuniquely advance medicine as it allows functionalizing the patches withmultiple ligands to target different types of cells. In biomedicalimaging, the patch cluster effect can significantly enhance the imagingsignal due to the high density of imaging molecules in a well-definedregion of the carrier.

Methods are known in the art for synthesizing patchy particles. Forexample, the synthesis of lipid polymeric patchy particles is describedin “Spontaneous Formation of Heterogeneous Patches on Polymer-LipidCore-shell Particle Surfaces during Self-Assembly”, Small, 2012. Theparticles can be synthesized using nanoprecipitation and emulsionmethods.

Particle formation depends on physical and mechanical parameters. Theshear stress that a polymer blend undergoes during the emulsificationstep in the particle's synthesis has been found to be an importantparameter for the formation of particles with patches. Thus, it isdesirable to evaluate the role of the shear stress in the formation ofthe internal and external morphology of lipid polymeric patchy particleswith single and multiple patches.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a lipid polymeric patchy particlethat includes a hollow core, a shell surrounding the hollow core and, asingle patch formed on the particle's shell; or a solid core, a shellsurrounding the solid core, and multiple patches formed on theparticle's shell.

At least one of the core and the shell can have formed thereon lipidstructures different from the one or more patches.

In certain embodiments, the lipid polymeric patchy particle includes oneor more biocompatible, biodegradable polymers. The one or more polymerscan be selected from the group consisting of sodium polystyrenesulfonate, polyethers, such as polyethylene oxide, polyoxyethyleneglycol and/or polyethylene glycol, polyethylene imine, a biodegradablepolymer such as polylactic acid, polycaprolactone, polyglycolic acid,poly(lactide-co-glycolide) polymer (PLGA), and copolymers, derivativesand mixtures thereof. A preferred polymer is PLGA. Further, the lipidpolymeric patchy particle can include a semi-conductor polymer, such as,poly [2,6-(4,4-bis-(2-ethylhexyl)-4H-cyclopenta[2,1-b;3,4-alt-4,7(2,1,3benzothiadiazole)] (PCPDTBT), which can form aninner lining in the particle core of particles with a hollow core or canbe embedded in particles with a solid core.

The lipid polymeric patchy particle can include one or morelipid-PEGylated-functional groups. For example, DSPE-PEG-R, wherein R isamino, methoxyl or maleimide.

The shell can include a first shell and a second shell. The first shellcan be formed by one type of LPFG (e.g., DSPE-PEG-NH₂) and the secondshell can be formed by a different LPFG (e.g., DSPE-PEG-maleimide).

The hollow core can include at least one payloads, such as, a drug, afluorescent dye and mixtures and combinations thereof.

In another aspect, the invention provides a method of synthesizing alipid polymeric patchy particle having a single patch or multiplepatches. The method includes dissolving the polymer in a first solventforming a first solution (often referred to as an oil phase); dissolvinglipid-PEGylated-functional groups in a second solvent (such as, water ora mixture of water and ethanol) forming a second solution (oftenreferred to as an aqueous phase); the first and second solutions forminga polymer blend; emulsifying the polymer blend using a high shear mixerassembly forming an emulsified blend; and evaporating the first andsecond solvents.

The first and second solvents can be different.

The particle can have a hollow core.

The morphology of the particle can be controlled by adjusting themagnitude of shear stress during the emulsification step.

High shear stress produces particles having a hollow core and a singlepatch.

Low shear stress produces particles having a solid core and multiplepatches.

The high shear mixer assembly can include a homogenizer workhead and arotor, and the magnitude of the shear stress can depend on size of a gapbetween an inner diameter of the homogenizer workhead and the outer edgeof the rotor. The gap can be from about 0.100 mm to about 0.127 mm.

The method can further include functionalizing the single patch or themultiple patches with a wide variety of organic and inorganicnanoparticles, such as, proteins and gold nanorods, respectively, toform a functionalized particle. The functionalized particle with goldnanorods can be employed in photoacoustic imaging.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an image that shows a lipid polymeric patchy particle having acore and a double shell.

FIG. 2 is a schematic showing a top view of a homogenizer's workhead androtor of a high shear mixer and a tubular mixing assembly.

FIG. 3 includes images (Views A through I) showing the external andinternal morphology of a lipid polymeric particle with multiple patchesand a solid core, in accordance with certain embodiments of theinvention.

FIG. 4 includes images (Views A, B, D and E) showing the external andinternal morphology of lipid polymeric particles, in accordance withcertain embodiments of the invention.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The invention relates to lipid polymeric patchy particles and, moreparticularly, to producing the particles having certain, e.g.,pre-selected, physicochemical properties, external and internalmorphologies and internal properties. Patchy particles with single andmultiple patches are natural photoacoustic and ultrasound agents. Theinvention also relates to methods and syntheses for producing the lipidpolymeric patchy particles, and mechanisms for controlling or tuning thephysicochemical properties, external and internal morphology andinternal properties of the particles. The particles can have one or morefunctionalized domains or patches that provide controllable particlebinding and orientation. Further, the size of the particles, number ofpatches and shells, as well as the thickness of the particle's shell canbe controlled or tuned in accordance with the invention. The externaland internal morphology and physicochemical properties of the particlesdepend on chemical, physical and thermodynamic factors. For example, thechemical factors can include the solvent-solvent, polymer-solvent andpolymer-polymer-solvent interactions.

Among physical or mechanical parameters, it has been found that highshear stress is the most important parameter for the formation of patchyparticles. In these circumstances, the shear stress overcomes thechemical molecular forces that drive the interaction between polymer,e.g., PLGA, lipid functional group(s), e.g., DSPE-PEG-R, and solvent.The patch(es) are formed instantaneously during an emulsification stepin the synthesis of the particles. The form of the patch depends on theamount of shear stress experienced by the polymer blend during theemulsification step. The amount of shear stress can vary. For example, ahomogenizer's workhead with a 1″ diameter used in the emulsificationstep of the particle's synthesis at 4000 rpm renders patch(es) that aremore bulky, e.g., the patch(es) protrude or appear to “stick on” to theparticle's shell. A homogenizer's workhead with a ⅝″ diameter used at4000 rm renders patch(es) that are not bulky, but rather part of thepatch is embedded in the polymer matrix.

The lipid polymeric patchy particles (e.g., nanoparticles ormicroparticles) generally have a core-shell structure, which has acentral core surrounded, or encapsulated, by a shell that forms an outersurface. In certain embodiments, the particle can have more than oneshell. The one or more shells can include a single lipid-containingpatch or multiple lipid-containing patches. A single patch or multiplepatches can be formed by lipid functional groups, e.g., LPFGs, on theparticle's shell(s). The functional groups may be arranged in one ormore of the particle's surface domains, e.g., patches, and shells, witheach domain including a majority of the same type of functional group.The functional groups provide surface chemistry and surfacephysicochemical properties.

The core can be hollow or solid. The hollow core can be filled with oneor more components, e.g., one or more “payloads”, and the solid core canhave the component, e.g., “payload”, embedded therein. Some particlescan have lipid-based structures entrapped in the particle's core and onthe surface. These lipid-based structures can function as enhancercontrast agents. In certain embodiments, the payload is a drug, afluorescent dye or a mixture or combination thereof.

The particles can be loaded with a wide variety of active agents, e.g.,therapeutic agents, for enhanced drug targeting and delivery, and orenhanced efficacy of the active ingredient. Thus, the particles arecapable of delivering active ingredients or biofunctional agents to oneor more different targets within a specific environment, e.g., the bodyof a human or animal subject, the skin, other organs (e.g., eye, heart,liver, pancreas, lungs and prostate).

The size of the particles can be between about 200 nm to about 10 um. Incertain embodiments, patchy nanoparticles of about 200 nm size aresynthesized by a mixture of DSPE-PEG-R and sodium dodecyl sulfate (SDS)at a concentration 8×10⁻³ M. SDS reduces the particle's size andimproves solubility. Nano-scale particles are generally considered to beup to 1000 nm at their largest cross-sectional dimension. Micron-scaleparticles are over 1.0 micron at their largest cross-sectional dimension(e.g., 1.0 micron up to 100 microns, or larger, e.g., 1.0 to 2.0microns, 1.0 to 10.0 microns, 5 to 25 microns, and 25 to 50 microns).

The patchy particles can be formed using various polymers known in theart. Preferred polymers are biocompatible, biodegradable and FDAapproved. These particles possess good blood biocompatibilityproperties. Generally, the particle design allows for functionalizingthe surface of polymeric particles, including lipid polymeric hybridparticles, PLGA, PLA and the like, with one or more different functionalgroups. This design enables the particles to bind to variouscombinations of different biomolecules, e.g., antibodies, proteins,peptides, aptamers, and the like.

In certain embodiments semi-conducting polymer can be used to form thepolymeric particles.

At least one phase can be designed to have one or more of the followingproperties based on the material selection: hydrophobic,positively-charged (cationic), negatively charged (anionic), andpolyethylene glycol (PEG)ylated.

In certain embodiments, the particles can be formed from two elements:(i) hydrophobic polymer, which forms the hydrophobic core that mayencapsulate a payload, and (ii) lipid functional group(s), which formone or more patches on the particle's core and/or shell. One end of thelipid binds to the shell of the core structure (and can extend outwardsfrom the core shell) and another opposite end includes the functionalgroup(s). The particles can include more than one lipid functionalgroup. For example, a first end of a first lipid can bind to the shellof the core structure and a second end includes a first functionalgroup, and a first end of a second lipid can bind to the shell of thecore structure and a second end includes a second functional group.

Suitable non-limiting polymers for use in the invention include sodiumpolystyrene sulfonate, polyethers, such as polyethylene oxide,polyoxyethylene glycol and/or polyethylene glycol, polyethylene imine, abiodegradable polymer such as polylactic acid, polycaprolactone,polyglycolic acid, poly(lactide-co-glycolide) polymer (PLGA), andcopolymers, derivatives and mixtures thereof.

A hydrophobic polymer for use in the invention can be selected fromthose known in the art, such as, but not limited to,poly(D,L-lactide-co-glycolide (PLGA). The lipid functional group(s) alsocan be selected from those known in the art, such as,lipid-PEGylated-functional groups (LPFGs). The LPFGs can include a lipidbound to polyethylene glycol (PEG) and a functional group bound to thePEG. Non-limiting examples of LPFGs include, but are not limited to,1,2-distearoyl-sn-glycerol-3-phosphoethanolamine (DSPE)-N-poly(ethyleneglycol) (PEG) with a terminal or functional group R: DSPE-PEG-R, where Ris amino, methoxyl, or maleimide, e.g., DSPE-PEG-NH₂, DSPE-PEG-OCH₃ andDSPE-PEG-MAL.

LPFGs can be used as building blocks for the synthesis of a wide varietyof materials, e.g., nanotherapeutics, because their end-terminalfunctional groups can be functionalized with a variety of organic and/orinorganic molecules. Furthermore, LPFGs offer the advantage ofsynthesizing multifunctional nanoparticles in which high control overthe number and ratio of functionalities on the particles' surface can beachieved without the need of orthogonal chemical reactions.

The LPFGs can have a different arrangement in the particle's surface ascompared to the edge and center of the patch. For example, thefunctional group of the DSPE-PEG-R molecule may stick out at the edge ofthe patch and shell, which allows functionalization with molecules, andthe LPFGs in the center of the patch may have a different arrangement.For example, the DSPE fragment of the DSPE-PEG-R may be exposed tofurther functionalization instead of having R attached thereto.

The formation of the patches on the particles resembles the phasesegregation phenomenon observed in polymer blends and lipid rafts.However, the polymer blend systems of the invention involve twodifferent polymers, e.g., PLGA and LPFG(s), and a tri-solventcomposition, e.g., water, ethanol and ethyl acetate. Therefore, withoutbeing bound by any particular theory, it is believed that the formationof patchy polymeric particles is due to the shear stress that thepolymer blend undergoes during the emulsification step.

In certain embodiments, two different LPFGs are employed to produceparticles having a double shell. Each of the shells having a differentsurface chemistry. FIG. 1 shows a particle having a core, a first shell(e.g., including a first LPFG) and a second shell (i.e., including adifferent second LPFG). The first shell can be formed by one type ofLPFG (e.g., DSPE-PEG-NH₂) and the second shell can be formed by adifferent LPFG (e.g., DSPE-PEG-maleimide).

In certain embodiments, good blood biocompatibility is achieved using aspecific LPFG, such as, DSPE-PEG-OCH₃, to synthesize patchy polymericparticles that likely can remain in circulation in a patient for atleast 48 hours, which extends the time available for medical imaging. Ithas been found that patchy polymeric particles synthesized withDSPE-PEG-folic acid and a mixture of DSPE-PEG-NH₂ and SDS at 8×10⁻³ Mcan remain in circulation for 24 hours in mice.

Lipid polymeric patchy particles can be prepared in general usingvarious known methods. For example, single-step nanoprecipitationmethods are described in U.S. Pat. No. 5,118,528, which is incorporatedherein by reference. These methods can be used to synthesizenanoparticles by mixing a solution containing a substance into anothersolution (i.e., a non-solvent) in which the substance has poorsolubility. For example, polymer (e.g., PLGA-PEG) nanoparticles can bemade in which polymer solutions in either water-immiscible orwater-miscible solvents are added to an aqueous fluid (i.e., thenon-solvent). Such nanoprecipitation methods are also described, forexample, in PCT WO 2007/150030.

Further, various methods are known to link or bind the lipid functionalgroup(s) to the core structure using covalent bonds or non-covalentbonds, such as, Van der Waals forces, to form the particles.

Polymeric- and lipid-containing particles can be made by (i) dissolvinga polymer in a volatile, water-miscible organic solvent to form a firstsolution (referred to as the oil phase); (ii) dissolving a plurality offirst and second amphiphilic components bound to heterofunctionallinkers in an aqueous solvent to form a second solution (referred to asthe aqueous phase), wherein the amphiphilic components each have ahydrophobic end and a hydrophilic end, the first heterofunctional linkereach includes a first functional group, and the second heterofunctionallinker each includes a second functional group, and (iii) combining thefirst and second solutions such that a polymeric nanoparticle is formedhaving a polymer core surrounded by amphiphilic components, wherein theheterofunctional linkers extend from the amphiphilic components and thefirst and second functional groups form an external mosaic of surfacedomains, each domain generally including a majority of one type offunctional group.

In certain embodiments, synthesis of the patchy particles includesnanoprecipitation and emulsification. Polymer and lipid solutions aremixed to form a polymer blend, which is subjected to high shear mixingto form an emulsified mixture. For example, an aqueous phase can beprepared by dissolving DSPE-PEG-R, e.g., DSPE-PEG-(2000)amine1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-2000 (DSPE-PEG-NH₂) or DSPE-PEG(2000) maleimide1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethyleneglycol)-2000 (DSPE-PEG-MAL) in solvent, e.g., ethanol and/or water. Thissolution is homogenized at, for example, 1000 rpm, for a period of time,e.g., 15 seconds. Polymer, e.g., PLGA solution (in ethyl acetate), isadded to the aqueous phase. The polymer blend is homogenized, forexample, at 2000 rpm or 4000 rpm for a period of time, e.g., 1 minute,using a high shear mixer and tubular mixing assembly, to produce anemulsified mixture. The solvent is then allowed to evaporate andparticles are formed.

It has been found that shear stress is a critical physical factor insynthesizing the patchy particles having different internal and externalmorphologies. The morphology of the core and the shell can depend on theshear stress applied to the polymer blend during synthesis of theparticles. Shear stress promotes and enhances mutual miscibility betweenPLGA and LPFGs. A sufficient amount of shear stress is necessary to formpatchy particles; that is, without enough shear stress, patchy particlescannot be formed. Further, the shear stress can be adjusted duringsynthesis to impart certain morphologies to the resulting particles. Theamount of shear stress exerted by the high shear mixer and tubularmixing assembly on the polymer solution during emulsification can impartdifferent morphology to the resultant particles.

Without intending to be bound by any particular theory, it is believedthat during the pre-emulsification step, the LPFGs form liposomes (e.g.,multilamellar and unilamellar vesicles) because the concentration ofLPFGs used in the particle's synthesis is above the critical micelleconcentration of DSPE-PEGm. However, because of the incorporation ofadditional ethanol, ethyl acetate and PLGA, the physical integrity ofthe liposomes is affected. When previously dissolved PLGA in ethylacetate is incorporated into the aqueous phase, a phase segregationphenomenon takes place initiated by the solvents (e.g., water-ethanoland ethyl acetate). The DSPE fragment of LPFGs interacts favorably andrapidly with the PLGA during the pre-emulsification step. The polymerblend undergoes a high shear stress during the emulsification step,which promotes the miscibility between PLGA and LPFGs giving rise to adroplet with a core-shell structure. The presence of high shear stressenhances the miscibility of the polymer blend inducing a pronouncedphase segregation phenomenon that results in the formation of thecore-shell structure followed by the formation of a patch. During thesechemical and physical conditions, droplets coated with LPFGs having anindentation are formed, and the remaining LPFGs start forming the patch.The patch may be formed by a pile-up of lipid-based structures. Thepatchy particles that result from this process have a hollow core, ashell formed by PLGA-DSPE-PEG-NH₂, and a patch formed by a pile-up ofLPFGs.

High shear stress of the polymer-lipid blend during synthesis of theparticles, forms particles having a hollow core and a singlelipid-containing patch. In contrast, low shear stress of thepolymer-lipid blend during synthesis of the particles, forms particleshaving a solid core and multiple lipid-containing patches. Further, thethickness of the particle's shell can be controlled by tuning the shearstress that the polymer blend undergoes during the emulsification stepof the particle's synthesis. In general, the higher the shear stress,the thicker the particle's shell. The thickness of the particle's shellcan be determined by cross-sectioning particles that have core-shell andpatch-core-shell structures.

Shear stress can depend on various factors, such as, the viscosity ofthe polymer blend solution and/or the dimension, e.g., diameter, andshape of the homogenizer workhead that is used to emulsify the polymerblend solution. The fluid mechanics as well as the fluid dynamics of thepolymer solution has an influence in the shear stress that the polymerblend undergoes during emulsification.

As aforementioned, a high shear mixer and tubular mixing assembly can beused to form an emulsified mixture from the polymer blend solution. Thisassembly includes a homogenizer having a workhead and a rotor. Varioushomogenizers and workhead designs are known in the art and may beemployed in the invention. In general, the homogenizer workhead is atubular structure including a hollow space or cavity formed by a wallhaving an inner diameter, an outer diameter. A plurality of shaped holesare formed in the perimeter of the wall of the tubular structure, e.g.,a screen, through which the polymer blend passes. The tubular structureis typically composed of metal and the holes can be round or square, andthe size, e.g., diameter, of the holes can vary. In certain embodiments,the homogenizer workhead can include round holes. Further, thehomogenizer workhead includes a rotor, e.g., blade, positioned withinthe hollow space of the tubular structure. In general, the homogenizerworkhead with the rotor therein, is inserted into a container, e.g.,beaker, holding the polymer blend. The polymer blend is forced into,e.g., enters, the cavity of the workhead with rotor therein, and thepolymer blend is discharged through the holes, e.g., screen.

FIG. 2 is a schematic showing an upper view of a homogenizer workheadhaving a rotor positioned therein. As shown in FIG. 2, there is a gapformed between the outer ends, e.g., outer edge(s), of the rotor and theinner surface or diameter of the homogenizer workhead, such that theouter ends of the rotor are not in contact with the inner surface ordiameter of the homogenizer workhead. The magnitude of the shear stressis determined by the size of the gap. The smaller the gap (the shorterthe distance between the outer ends of the rotor and the inner surfaceor diameter of the homogenizer workhead), the higher the shear stressthat the polymer blend solution undergoes during emulsification in theparticle's synthesis. In certain embodiments, the gap can be from about0.100 mm to about 0.127 mm or from about 0.100 mm to about 0.137 mm. Ithas been determined that a gap of 0.137 mm corresponds to a shear stressof about 450 to 600 dynes/cm².

Shear stress can be calculated based on the following formula:

$\tau = {{- V_{t}}\mu \frac{2}{d}}$

wherein,

τ=shear stress;

V_(t)=tangential velocity;

μ=viscosity of the polymer blend solution;

d=gap size (distance or difference between the inner diameter of thehomogenizer's workhead and the outer end(s)/edge(s) of the rotor); and

V_(t)=ωr

wherein,

ω=2πf;

frequency=shear rate=revolution per minute;

π=3.1616; and

r=homogenizer's workhead radius.

High shear stress during emulsification of the polymer blend formsparticles with a single patch and a hollow core because the shear stressforce overcomes the van der Waals interaction between DSPE-PEG-R andPLGA. The patchy particles resulting from high shear emulsification havea hollow core, a shell formed, for example, by PLGA-DSPE-PEG-R, and asingle patch formed by a pile-up of LPFGs. In contrast, low shear stressduring emulsification of the polymer blend forms particles with multiplepatches and a solid core because the van der Waals interaction betweenDSPE-PEG-R and PLGA is strong. With low shear stress, LPFGs form a thinshell on the polymeric core. When the shear stress force is low and thevan der Waals interaction between DSPE-PEG-R and PLGA is strong,lipid-based structures may be formed, e.g., entrapped or embedded, intothe solid core of the particles. Without intending to be bound by anyparticular theory, it is believed that the lipid-based structuresinclude a particular arrangement of lipid-polymer based functionalgroup. The lipid-based structures may also form on the shell.

Controlling or tuning the amount or degree of shear stress duringparticle synthesis can determine the morphology of the particle, i.e.,core-shell and core-shell-patch, and the thickness of the shell.

FIG. 3 shows images of a lipid polymeric particle with multiple patchesformed in accordance with certain embodiments of the present invention.View A is an image showing the external morphology of the particleincluding multiple patches, e.g., LPFGs. Views B through I are images ofthe internal morphology of the particle based on cross sections of thesame particle at different or varying depths. View B shows a solid coreand Views C-I show lipid-based structures that are formed, e.g.,entrapped or embedded, in the solid core. In particular, View F is aclose-up of the lipid-based structures embedded in the particle core;View G shows an arrangement of lipid-based structures near a patch; ViewH shows the half of the particle's core completely covered bylipid-based structures; and View I is a close-up of the lipid-basedstructures near a patch. Based on these images, it is evident that thelipid-based structures found in the particle's core have a differentarrangement or configuration from that of LPFGs in the patch.

FIG. 4 shows images of lipid polymeric particles with different internaland external structures. View A is an image of the external morphologyof a particle having a single patch and View B is a cross-section of theinternal morphology of the same particle having a hollow core. View D isan image of the external morphology of a particle having multiplepatches and View E is a cross-section of the internal morphology of thesame particle having a solid core and lipid-polymer-based structuresentrapped in the core.

As mentioned herein, patchy particles having surface domains or patchesprovide advantages in a wide variety of fields and applications. Suchapplications include imaging applications because the clusters on theparticle's surface can enhance the imaging signal. This effect willreduce toxicity and maximize the imaging effect. It was found that theabsorbance of particles that have a patch-core-shell structure is 1.5times higher than particles that have only a core-shell structure. Theabsorbance of particles that have two cores and two patches has threetimes the absorbance of core-shell structure and twice the absorbance ofthe single patch-core-shell structure. The higher the absorbance, thehigher the amount of light absorbed by the particle. This highabsorption property of these patchy particles is advantageous forphotoacoustic and ultrasound imaging where the adsorbed light isconverted into heat leading to a transient thermoelastic expansion andultrasonic emission, which will form the image.

Controlling the thickness of the particle's shell is useful forphotoacoustic and ultrasound applications because thin shells can bedisrupted easily with light. The methods of the invention provide thecapability of synthesizing patchy particles with very thin shells.

Further, semi-conductor polymer, such as, poly[2,6-(4,4-bis-(2-ethylhexyl)-4H-cyclopenta[2, 1-b;3,4-alt-4,7(2,1,3benzothiadiazole)] (PCPDTBT), can be incorporatedduring synthesis, e.g., in the polymer blend. The semi-conductor polymercan form an inner lining in the particle core of particles with a hollowcore or can be embedded in particles with a solid core. That is, patchyparticles having an interlining are produced when the shear stress ishigh or patchy particles having the semiconducting polymer embedded inthe polymer matrix are produced when the shear stress is low. Thesetypes of the arrangement of the semiconducting polymer can beadvantageous for use in photoacoustic applications.

Polymeric particles with multiple patches are unique materials becausetheir external surface is different from their internal morphology,which provides several advantages. For example, the outer surface hasmultiple domains that can be further functionalized with a variety oforganic or inorganic molecules, e.g., nanoparticles, such as, but notlimited to proteins and gold nanorods, respectively. The polymerparticles functionalized with gold nanorods can be employed for imagingpurposes because these particles emit a photoacoustic (PA) signal.

In certain embodiments, LPFGs form a patch on a lipid polymeric particleand a portion of the LPFGs that form the patch are functionalized withgold nanorods. The R fragment of DSPE-PEG-R protrudes, e.g.,“sticks-out”, such that it may be functionalized. The LPFGs that formthe center of the patch may have a different arrangement from the oneobserved at the edge of the patch. Gold nanorods have high attenuationcoefficient. The thinner the particles' shell, the less is theattenuation coefficient and the higher the adsorption and thermalexpansion. Therefore, the higher is the resolution of the image obtainedusing the photoacoustic method.

The lipid polymer patchy particles combine characteristics of bothliposomes and polymer particles, and are able to carry poorly solublematerials, e.g., drugs. Their circulation half-life is longer than thatof polymeric particles, and slightly shorter than that of liposomes.These particles have multivalent targeting abilities and can be designedto provide a sustained and/or controlled drug release.

Furthermore, the patchy particles can encapsulate a wide range ofpesticides, such as atrazine, which is the most widely used herbicide inthe market. Also, at the same time, through the patch there can bereleased growth factor that can promote and enhance the growth of theplant. Particles with multiple patches can be used as carriers formultiple-growth factor clusters. Particles with two patches and twocores can release a pesticide through the core and release two differentgrowth factors through the patches and shells.

It will be appreciated by those skilled in the art that changes could bemade to the embodiments described above without departing from the broadinventive concept thereof. It is understood, therefore, that thisinvention is not limited to the particular embodiments disclosed and thefollowing examples conducted, but it is intended to cover modificationsthat are within the spirit and scope of the invention.

EXAMPLES

Solvents used in the examples were purchased at analytical grade fromSigma-Aldrich (St. Louis, Mo.). DSPE and PEG-based polymers werepurchased from Avanti Polar Lipids (Alabaster, Ala.) and Laysan Bio(Arab, Ala.). PLGA was purchased from Lactel (Pelham, Ala.).

Example 1—Patchy Particle's Internal Structure

To further understand the role of the shear stress in patchy particleformation, particles at different shear stress rates were synthesized.It was found that multiple and single patches were formed at 2000 rpmand 4000 rpm, respectively. Previously, by cross sectioning thesingle-patch particles, it was found that these particles have a hollowcore. Using the same rationale, a cross-section was taken of theparticles with multiple patches. The cross-sections revealed that theseparticles had a solid core with entrapped lipid-based structures. Uponcross-sectioning of the particle's core, it was observed that thelipid-based structures were embedded in the entire polymer matrix.Moreover, the lipid-based structures were only observed in theparticle's core and not in the patch or patches. This finding suggestedthat although the lipid-based structures in the particles' core weremade of LPFG, their arrangement was different from that in patch orpatches. In fact, the lipid-based structures in the particle's coretended to separate from the patch or patches.

Computational Fluid Dynamic (CFD) simulations showed that the wall shearstress at 2000 rpm was very low compared with that at 4000 rpm. Based onthese results, it was evident that the shear stress rate isdeterminative of whether a particle's internal structure is solid orhollow.

Molecular Dynamics (MD) simulations were developed to evaluate theself-assembly process of PLGA and LPFG. Due to the high number of atomsneeded to develop a MD of a microparticle of an average size of 2 μm indiameter, it was necessary to perform MD of a nanoparticle of 18 nm indiameter. This nanoparticle was made of poly (lactic-co-glycolic acid(PLGA)), a biocompatible, biodegradable and FDA-approved polymer. Thetwo monomers of PLGA: lactic acid and glycolic acid were built and laterreplicated at random, making a total of 419 lactic acid (76%) and 131glycolic acid (24%) monomers for a single chain of PLGA.

Full atomistic MD simulation was carried out to elucidate the nature ofthe interactions that drive the formation of DSPE-PEG-NH₂/PLGA complex.To achieve this goal, pair interaction energy values were obtained fromDSPE (tails)-PLGA, PEG-PLGA and PEG-DSPE (tails) fragments, of the samenumber of atoms, taking into account the last part of the simulation.This calculation was performed using NAMD Energy plugin included inVIVID software. As hypothesized, the van der Waals energy between thepolymers was more relevant than the electrostatic energy. MD revealedthat the most favorable van der Waals interaction occurred between DSPEand PLGA as the interaction between the DSPE and PEG sections wasdisplaced. The DSPE-PEG interaction was more relevant at the beginningof MD but that interaction changed as time passed. Similarly, PEG andPLGA also showed a favorable van der Waals interaction but not as strongas DSPE-PEG. Furthermore, as was observed from a radial distributionfunction (RDF), DSPE tails were partially embedded into PLGAnanoparticle, while some sections were interacting with PEG polymers.PEG section was also in part buried into PLGA. DSPE appeared as aninterface between PEG and PLGA, while PEG tended to displace moretowards the surface. At the same time, the calculation of solventaccessible surface area (SASA) ratified that DSPE-PEG remained moreprotected from the environment (lower values of SASA) being in contactwith PLGA polymers. It was evident how PLGA and DSPE-PEG-NH₂ interactedto each other between the hydrophobic regions. Knowing the interactionbetween PLGA and DSPE-PEG-R contributes to acquiring more control on theparticle's surface chemistry, and therefore assists in improving theirclinical performance, as the particle's surface chemistry plays animportant role in the protein-corona effect. Moreover, use of the MDsimulations can vary the PEG's molecular weight and molar ratio withrespect to the PLGA to predict the particle's surface chemistry.

Coarse-grained MD were developed to understand the formation of thehollow aspect of the particle's core. In this type of simulation, avariable pressure was incorporated to emulate the shear stress that thepolymer blend undergoes during the emulsification step of the particle'ssynthesis. To start the simulation, DSPE-PEG polymers were placed atrandom, in a water box, on the top of the PLGA nanoparticle (FIG. 3bia). As the simulation continued, hydrophobic DSPE tails started toagglomerate, while water penetrated both PEG sections and PLGA block.Towards the end of the simulation, there was observed the formation ofbilayers which were composed by DSPE molecules, while DSPE-PEG polymerscollapsed into the surface of the PLGA nanoparticle due to the effectsof the high pressure applied to the system. Analysis of the systemdensity over the last frame of the simulation showed that PLGA densityencompassed the area 0 nm<z<15 nm. The density of the nanoparticle didnot constitute a rigid core. Specifically, the area where the densitydecayed (3 and 8 nm in the z-axis) coincided with an increase in thedensity of water in that zone. Moreover the fact that the density ofwater is >0 along the block demonstrated that water can penetrate PLGAand PEG sections. As a result of this phenomenon, the nanoparticleformed by the PLGA polymer showed some cavities inside, denoting theentry of water. Experimentally, it was found that particles with singlepatches have a hollow core. Initially, it was hypothesized that theparticle's hollow core was formed because the shear stress force thatthe polymer blend undergoes during the emulsification step in theparticles synthesis, overcomes the van der Waals interaction that existsbetween DSPE-PEG-R and PLGA. Although the cavity of the particles wasobserved in MD when the nanoparticle's core was already formed, it wasevident that the pressure parameter incorporated into the MDS or theshear stress force applied during the emulsion of the polymers causedthe particle's hollowness.

Example 2—Particle's External Structure

MD was also employed to provide information about the arrangement of theLPFGs in the particle's surface. These MD showed that the hydrophobictails (DSPE) distribution displayed regular peaks, indicating theformation of bilayers along the block. A single peak of DSPE was nearthe surface of PLGA nanoparticle (z=12), showing tails that collapseinto its surface. PEG sections were distributed in the same area asDSPE, 5 nm<z<35 nm. Where the density of PEG decayed, the density ofwater increased, showing that this polymer was acting as a sponge. Infact, the ground section of the block, encompassing the area 0<z<13, wassimilar as compared with the nanoparticle formed along the full-atommolecular dynamics simulation. In this area, PEG and PLGA competed inthe interaction with DSPE tails.

To facilitate the analysis of the bilayers formed during thecoarse-grained simulation, there was selected a small block of DSPE-PEGpolymers from the final aggregate. As was deduced from density profile,DSPE tails occupied the area 5 nm<z<11 nm. DSPE head groups (defined by+NH₃ ⁺ groups) represented by two peaks (purple line), in the region 4-6nm and 10-12 nm, denoted the interfacial area of the lipids, showing thetypical profile of a bilayer. PEG polymers were distributed in the upperand lower part of the bilayer, which decreased their density in the DSPEbilayer area. A schematic representation of the arrangement ofDSPE-PEG-NH₂ on the particle's surface was observed. MD simulationsconfirmed that DSPE fragment of the LPFGs formed not only the interfacebetween PLGA and DSPE-PEG-NH₂ but also the formation of a bilayer in theparticle's surface. Moreover, MDS showed that part of DSPE and PEG areburied in PLGA. The structural information about the arrangement of theLPFG in the particle's surface was necessary to achieve high control onpatchy particles' surface chemistry. This information also may berelevant for the selection of the payloads for a particular application.

The MDs showed that some molecules of H₂O were also entrapped in theparticle's core whether the core was solid or hollow. Because of theeffect of the pressure, and due to the water being incompressible,several holes were formed in the PLGA nanoparticle. The lipid andpolymer molecules offered more interstice that may experience morecompression than bulk water. When the shear stress force was low, thevan der Waals interaction between PLGA and DSPE-PEG-NH₂ was strong,leading to the formation of a solid particle's core with entrappedlipid-based structures. The lipid-based structures formed in theparticle's core were presumably a particular arrangement oflipid-polymer based functional group. MD showed how the DSPE fragment ofthe DSPE-PEG-NH₂ interacted closely and preferentially with the lacticmonomer, which was not unexpected because the DSPE fragment and lacticacid are hydrophobic. Moreover, in the presence of low shear stress manyDSPE-PEG-NH₂ got entrapped in the particle's core because of the highdiffusion coefficient of the DSPE fragment that enhanced and facilitatedits interaction and penetration into the PLGA copolymer.

MD simulations demonstrated the formation of a lipid bilayer in theparticle's shell formed by DSPE-PEG-R, which is an important findingbecause it provides insights about the particle's surface. Therefore,the interaction of the nanocarrier with proteins and cells could bepredicted. The fact that there is a lipid-bilayer in the particle'ssurface allows for incorporation of hydrophobic molecules between thebilayer. Also, MD simulations corroborate the experimental finding thathigh shear stress produces particles with a single and hollow core.

Example 3—Biomedical Applications of Patchy Polymeric Particles

It was found that the patchy polymeric particles possess unique opticalproperties. They induced a significant PA signal, which wasdose-dependent, in the near-infrared (NIR) region (i.e, 600-950 nm).Clinically, it was known that the NIR region (700-1100 nm) is where theinfluence of the main tissue absorbing components, oxy-anddeoxyhemoglobin (max<600 nm) as well as water (max>1150 nm) is minimal.Therefore, this region of the spectrum was considered as the idealoptimal imaging window. Particles with multiple patches induced a higherPA signal than patchy particles with single patches. The PA signalinduced by particles with multiple patches and solid core wasapproximately 9 times higher than the one emitted by particles withsingle patches and hollow core when the particle's concentration was 5mg/ml. This phenomenon was likely due to the ability of particles withmultiple patches to absorb more light than single patch-particles. Fromthe molecular level perspective, it is believed that particles withmultiple patches are better natural photoacoustic contrast agentsbecause they can offer a larger surface area to the solvent of theself-assembly process that generate the multiple patches.

Differential Scanning calorimetry was employed to evaluate the thermalproperties of these particles. Both particles exhibited a relativelysimilar melting point temperature. The melting temperature of patchyparticles with multiple patches was 59° C. and the melting pointtemperature for particles with a single patch was 57° C. There were onlytwo degrees of difference between them. Furthermore, single-patchparticles had a crystallization temperature (Tc) of approximately 2.2°C. while the multiple domain patchy particles had a Tc of approximately−5° C. The one sharp peak for each sample indicated that these sampleswere pure. Since the peaks were not broad, both samples were notpartially crystalline polymers but were amorphous. Since both samplescontained no a-nuclei, they had no crystallization peak and no a-meltingpeak. The baseline shifted lower towards the endothermic direction afterthe peak for both samples due to the increased heat capacity of thesample. The most significant difference between particles with singleand multiple patches was the enthalpy, the area under their peaks.Particles with multiple patches exhibited higher enthalpy than that ofparticles with single patches, which means that the former absorbed moreheat than particles with individual patches. This thermal profilecorrelated well with the intrinsic photoacoustic properties of patchyparticles. High enthalpy seemed to render higher photoacoustic signal.It was attempted to enhance or modulate their PA signal byfunctionalizing their patch or patches with exogenous PA contrastagents, such as, gold nanorods and nanoshells or by tuning theirinternal properties. It is known to use PLGA particles as carriers forcontrast agents. The multiple patches can play an unparalleled imagingperformance because of the cluster effect induced by the patch.

Results and Conclusions of Examples 1-3

There was evaluated the self-assembly process involved in the formationof PLGA particles with single and multiple patches which are formed bythe unique arrangement of LPFGs. It was found that in the presence of ahigh shear stress force, particles with a single patch and with a hollowcore are formed because the shear stress force overcomes the van derWaals interaction between DSPE-PEG-R and PLGA. Particles with multiplepatches and solid core are formed when the shear stress is low, andtherefore the van der Waals interaction between DSPE-PEG-R and PLGA isstrong. Thus, the shear stress determines both the internal and externalmorphology of PLGA particles as well as their unique natural PAproperties. Additionally, MD revealed the formation of a lipid bilayeron the particle's surface.

Example 4—Synthesis of Lipid Polymeric Patchy Particles

Particles with single and multiple patches were prepared by asingle-emulsion method as reported in Salvador-Morales and Rasheed etal. Briefly, the aqueous phase of the mixture was prepared by dissolvingDSPE-PEG(2000) amine1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-2000 (DSPE-PEG-NH₂) or DSPE-PEG(2000) maleimide1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethyleneglycol)-2000 (DSPE-PEG-MAL) in 4% ethanol to a concentration of 1 mg/ml.To this solution, 6 mL of 4% ethanol was added, and the solution washomogenized at 1000 rpm for 15 s. 4 mL of PLGA solution (15 mg/mL inethyl acetate) was immediately added to the aqueous phase. The mixturewas homogenized at 2000 rpm or 4000 rpm for 1 min using a L5M-A highshear mixer and ⅝ in. tubular mixing assembly. Fifty mL of deionizedwater was added dropwise to the emulsified mixture, and the volatilesolvent was allowed to evaporate overnight. Particles were centrifugedat 2000 rpm for 10 minutes using Millipore Amicon Ultra centrifugalfilter units with a MWCO of 100 kDa to remove completely the remainingsolvent and washed three times with deionized water. Subsequently,particles were examined with a FE-SEM (Zeiss) operating at 1.00 kVaccelerating voltage to visualize the particle's surface. The aqueousphase of particles with multiple patches was prepared with 1 ml ofDSPE-PEG-NH₂ and 1 ml DSPE-PEG-MAL. DSPE-PEG-NH₂ was labeled with Alexa594 (5 ug) before being mixed with DSPE-PEG-MAL.

Example 5—Focused-Ion Beam and Cross Sections

Patchy polymeric microparticles were cross-sectioned using a FEI FocusedIon Beam (FIB) with a gallium ion source operated at 1 kV, SE mode, and25000× magnification. Patchy microparticles were previously coated withgold-palladium alloy to protect particles from the ion beam.

Example 6—Computational Fluid Dynamics Simulations

To demonstrate the dependence of the fluid shear stress on the size ofthe gap between the inner diameter of the homogenizer tubular assembly'sworkhead and the rotor shaft, computational fluid dynamics (CFD)simulations were carried out for a gap size of 0.137 mm. The numericalsolutions of 3D incompressible Navier-Stokes equations were obtainedwith an edge based finite element solver. Unstructured grids composed oftetrahedral elements were generated and locally refined near the innerwall to obtain at least two points within the gap. The resulting meshhad approximately 3 million elements. The rotating piece was modeledusing immersed boundary methods based on unstructured grids. The rotorshaft was set in rigid-body rotation around its axis at 4000 rpm and2000 rpm. No slip boundary conditions were applied at all body surfaces,including the rotating rotor shaft. The polymer fluid density was set to1.0 g/cm³ and the viscosity to 0.031 dyn s/cm². The viscosity of thepolymer solution was measured using a MCR702 rheometer (Anton Paar GmbH)with a double-gap configuration. An explicit three-stage Runge-Kuttascheme with CFL=0.6 and a maximum time step of 5×10⁻⁵ was used toadvance the flow solution. All simulations were carried out in parallelon shared memory computers using OpenMP and were run on 16 processors.Results were saved at 1.5×10⁻⁴ s intervals and animations of the wallshear stress in the gap region were created.

Example 7—Hollow Patchy Polymeric Particle Synthesis

Hollow patchy polymeric particles were synthesized with differentworkhead dimensions including ⅜″, ⅝″ and 1″. The 1″ workhead with asquare screen head produced particles with “bulky patches” (see FIG.1EX). These particles had a hollow core and very thin shell ofapproximately 2 nm in thickness (see FIG. 2EX). The particlessynthesized with 1″ workhead were synthesized at different angularspeeds (rpm) ranging from 2000 rpm to 10000 rpm. When scaling up theparticle synthesis, the size of the patch of particles synthesized with⅝″, ⅜″ and 1″ workheads depended on the emulsification time and theangular speed. This was because a high volume of polymer solutionrequired more “mechanical energy” to emulsify the polymer solution thanthat required to emulsify a low volume of polymer solution. For example,when the volume of the polymer solution was 72 ml, the largest patcheswere formed when the polymer solution is emulsified at 8000 rpm at 4 minusing the 1″ workhead. As the volume of the polymer solution wasincreased, the emulsification time needed to be increased.

Patchy particles synthesized with 1″ and ⅝″ workheads had a high opticaldensity at different wavelengths. The optical density of these patchyparticles was approximately three times higher than the optical densityof gold nanorods which have been used as standard contrast agents inphotoacoustics. These patchy particles displayed high optical densityeven at 800 and 900 nm, whereas the optical density displayed by goldnanorods is typically very low at these wavelengths. Because theabsorption spectrum of these particles was high in the range of 600 to900 nm, these particles could perform exceptionally as contrast agentsfor photoacoustics and ultrasound applications. In addition, patchyparticles synthesized with the 1″ workhead had a very thin shell, whichprovides the potential to break up that shell with an external stimulus,such as, a laser. This peculiar feature of these particles may beadvantageous for a medical treatment wherein it is required that thedrug be released completely at once. Thus, these patchy particles may beideal carriers for a theranostic application where both imaging andtherapeutic effects are provided simultaneously.

1-18. (canceled)
 19. A lipid polymeric patchy particle comprising: (a) asolid core having an outer surface surrounding the solid core; (b) oneor more shells surrounding the outer surface; (c) a plurality of patchesformed on the outermost shell; and (d) a first lipid functional grouphaving a first end and a second end, wherein the first end is bound tothe outer surface, thereby forming a first shell, and wherein the secondend has a first functional group, wherein the outer surface comprises ahydrophobic polymer.
 20. The particle of claim 19, wherein the particlefurther comprises a second lipid functional group having a first end anda second end, wherein the first end of the second lipid functional groupis bound to the outer surface, thereby forming the first shell, togetherwith the first lipid functional group, and wherein the second end of thesecond lipid functional group has a second functional group, wherein thefirst lipid functional group and the second lipid functional group aredifferent.
 21. The particle of claim 19, wherein the hydrophobic polymeris a biocompatible, biodegradable polymer.
 22. The particle of claim 19,wherein the hydrophobic polymer is poly(lactide-co-glycolide) polymer.23. The particle of claim 19, wherein the first lipid functional groupis a lipid-PEGylated functional group.
 24. The particle of claim 23,wherein the first lipid functional group has a formula:-1,2-distearoyl-sn-glycerol-3-phosphoethanolamine (DSPE)-N-poly(ethyleneglycol) (PEG)-R, wherein R is a functional group.
 25. The particle ofclaim 23, wherein the first lipid functional group has a formula:-DSPE-PEG-R, wherein R is selected from amino, methoxyl, and maleimide(MAL).
 26. The particle of claim 19, wherein the particle comprises thefirst shell and a second shell, wherein the second shell surrounds thefirst shell.
 27. The particle of claim 26, wherein the particle furthercomprises a second lipid functional group having a first end and asecond end, wherein the first end of the second lipid functional groupis bound to the second shell, and wherein the second end of the secondlipid functional group has a second functional group, wherein the firstlipid functional group and the second lipid functional group aredifferent.
 28. The particle of claim 27, wherein the second lipidfunctional group has a formula -DSPE-PEG-NH₂ and wherein the differentlipid functional group has a formula -DSPE-PEG-MAL.
 29. The particle ofclaim 19, wherein bound is via covalent bonds.
 30. The particle of claim19, wherein a semi-conductor polymer is embedded in the solid core. 31.The particle of claim 30, wherein the semi-conductor polymer is poly[2,6-(4,4-bis-(2-ethylhexyl)-4H-cyclopenta[2,1-b, 3, 4-alt-4,7(2, 1,3-benzothiadiazole)] (PCPDTBT).
 32. The particle of claim 19, wherein apayload is embedded in the solid core.
 33. A method of making a lipidpolymeric patchy particle, wherein the method comprises: (a) combining:(i) a first solution comprising a lipid-PEGylated functional group; and(ii) a second solution comprising a hydrophobic polymer, thereby forminga polymer blend; (b) emulsifying the polymer blend using a high shearmixer assembly, thereby forming an emulsified blend, wherein theparticle comprises a hollow core and a single patch or wherein theparticle comprises a solid core and a plurality of patches.
 34. Themethod of claim 33, wherein the method further comprises adjusting themagnitude of shear stress, thereby controlling the particle's externaland internal morphology.
 35. The method of claim 34, wherein a highshear stress is applied and wherein the particle comprises the hollowcore and the single patch.
 36. The method of claim 34, wherein a lowshear stress is applied and wherein the particle comprises the solidcore and the plurality of patches.
 37. The method of claim 33, whereinthe high shear mixer assembly comprises: (a) a homogenizer workheadhaving an inner diameter; and (b) a rotor shaft having an outer edge,wherein the magnitude of shear stress is controlled by the distancebetween the inner diameter of the homogenizer workhead and the outeredge of the rotor shaft.
 38. The method of claim 33, wherein thedistance between the inner diameter of the homogenizer workhead and theouter edge of the rotor shaft is in the order of micrometers.